A case study in Stereocaulon

Received: 31 January 2017

|

Revised: 3 June 2018

|

Accepted: 5 June 2018

DOI: 10.1111/mec.14764

ORIGINAL ARTICLE

The complexity of symbiotic interactions influences the ecological amplitude of the host: A case study in Stereocaulon (lichenized Ascomycota) Lucie Vančurová1

| Lucia Muggia2

| Ondřej Peksa3 | Tereza Řídká1 |

Pavel Škaloud1 1 Faculty of Science, Department of Botany, Charles University, Prague 2, Czech Republic

Abstract Symbiosis plays a fundamental role in nature. Lichens are among the best known,

2

Department of Life Sciences, University of Trieste, Trieste, Italy 3 The West Bohemian Museum in Pilsen, Plzeň, Czech Republic

Correspondence Lucie Vančurová, Faculty of Science, Department of Botany, Charles University, Benátská 2, 128 01 Prague 2, Czech Republic. Email: [email protected] Funding information Czech Science Foundation, Grant/Award Number: GP13-39185P; Primus Research Programme, Grant/Award Number: SCI/13

globally distributed symbiotic systems whose ecology is shaped by the requirements of all symbionts forming the holobiont. The widespread lichen‐forming fungal genus Stereocaulon provides a suitable model to study the ecology of microscopic green algal symbionts (i.e., phycobionts) within the lichen symbiosis. We analysed 282 Stereocaulon specimens, collected in diverse habitats worldwide, using the algal ITS rDNA and actin gene sequences and fungal ITS rDNA sequences. Phylogenetic analyses revealed a great diversity among the predominant phycobionts. The algal genus Asterochloris (Trebouxiophyceae) was recovered in most sampled thalli, but two additional genera, Vulcanochloris and Chloroidium, were also found. We used variation‐partitioning analyses to investigate the effects of climatic conditions, substrate/ habitat characteristic, spatial distribution and mycobionts on phycobiont distribution. Based on an analogy, we examined the effects of climate, substrate/habitat, spatial distribution and phycobionts on mycobiont distribution. According to our analyses, the distribution of phycobionts is primarily driven by mycobionts and vice versa. Specificity and selectivity of both partners, as well as their ecological requirements and the width of their niches, vary significantly among the species‐level lineages. We demonstrated that species‐level lineages, which accept more symbiotic partners, have wider climatic niches, overlapping with the niches of their partners. Furthermore, the survival of lichens on substrates with high concentrations of heavy metals appears to be supported by their association with toxicity‐tolerant phycobionts. In general, low specificity towards phycobionts allows the host to associate with ecologically diversified algae, thereby broadening its ecological amplitude. KEYWORDS

diversity, ecological niches, lichen, phycobiont, specificity, symbiosis

1 | INTRODUCTION

hosts, enabling them to colonize habitats where they would normally not survive (Paracer & Ahmadjian, 2000). Lichens are an iconic

A number of invertebrates, such as sea anemones, corals and platy-

example of symbiotic systems, composed of various heterotrophic

helminths, as well as protists, have evolved mutualistic associations

and autotrophic organisms. The exclusive presence of multiple auto-

with photosynthetic partners. They provide photoassimilates to the

trophic and heterotrophic symbionts gives rise to a thallus with a

3016

|

© 2018 John Wiley & Sons Ltd

wileyonlinelibrary.com/journal/mec

Molecular Ecology. 2018;27:3016–3033.

VANČUROVÁ

|

ET AL.

3017

typical phenotype and a characteristic combination of secondary

pioneer lichens that grow in harsh conditions on bare substrates,

compounds (Spribille et al., 2016). Lichens are found in a wide range

such as lava flows and relatively exposed siliceous blocks, thereby

of terrestrial environments throughout the world. In some ecosys-

contributing to their weathering (Meunier, Kirman, Strasberg,

tems, lichens are the dominant autotrophs (Romeike, Friedl, Helms,

Grauby, & Dussouillez, 2014; Stretch & Viles, 2002). Multiple lin-

& Ott, 2002).

eages of Asterochloris are associated with diverse Stereocaulon spe-

Approximately 100 species within 40 genera of green algae and

cies (Nelsen & Gargas, 2008; Peksa & Škaloud, 2011). Chloroidium

cyanobacteria have been reported for the more than 20,000 species

was found in Stereocaulon nanodes (Beck, 2002), and members of the

of mycobionts (Kirk, Cannon, Minter, & Stalpers, 2008). The most

newly described genus Vulcanochloris are the phycobionts of Stereo-

common photobionts comprise the green algal genera Trebouxia and

caulon vesuvianum (Vančurová, Peksa, Němcová, & Škaloud, 2015).

Trentepohlia and the cyanobacterium Nostoc (Friedl & Büdel, 2008;

Previous ecological studies on lichen phycobionts focused mainly

Tschermak‐Woess, 1988b). The degree of specificity and selectivity

on the type of growth substrate (Bačkor et al., 2010; Leavitt et al.,

that both the fungal and algal partners show for each other is crucial

2013; Muggia et al., 2014). Several studies have investigated the

for the development of the lichen thallus. The term specificity delim-

effects of various climatic conditions (Fernández‐Mendoza et al.,

its the taxonomic range of acceptable partners, whereas selectivity

2011; Grande et al., 2017; Leavitt et al., 2016; Marini, Nascimbene,

refers to the preference for a certain group of partners (Rambold,

& Nimis, 2011; Peksa & Škaloud, 2011; G. Singh et al., 2017). As of

Friedl, & Beck, 1998; Yahr, Vilgalys, & Depriest, 2004; Yahr, Vilgalys,

late, Rolshausen, Dal Grande, Sadowska‐Deś, Otte, and Schmitt

& DePriest, 2006). Most mycobiont species associate with several

(2017) described mutualist‐mediated climatic niche expansion. More-

lineages of a single algal genus, frequently Trebouxia (Casano et al.,

over, global climate change events have also been discussed in asso-

2011; Helms, Friedl, Rambold, & Mayrhofer, 2001; Leavitt et al.,

ciation with lichen phycobionts. Aptroot and van Herk (2007)

2015, 2016; Leavitt, Nelsen, Lumbsch, Johnson, & St. Clair, 2013;

considered the genus Trentepohlia, whose members prefer warm and

Muggia, Perez‐Ortega, Kopun, Zellnig, & Grube, 2014; Nyati, Bhat-

humid climates, to be an indicator of climate change in temperate

tacharya, Werth, & Honegger, 2013; G. Singh et al., 2017). Zoller

zones. Most analogous studies, which considered the effects of tem-

and Lutzoni (2003) studied the interaction of basidiolichen Ompha-

perature on coral‐algae symbiosis, showed that the preferences for

lina with only one species of the genus Coccomyxa. The phycobiont

certain photobionts are key factors in the distribution of the host

diversity of the lichen‐forming fungal genera Cladonia (Bačkor, Peksa,

(Howells et al., 2012).

Škaloud, & Bačkorová, 2010; Beiggi & Piercey‐Normore, 2007; Pier-

As host distribution may be greatly influenced by the require-

cey‐Normore & DePriest, 2001; Škaloud & Peksa, 2010; Yahr et al.,

ments of the photobionts, the aim of our work was to determine the

2004) and Lepraria (Nelsen & Gargas, 2006, 2008; Peksa & Škaloud,

phycobiont (i.e., green eukaryotic photobiont) diversity of Stereo-

2011; Škaloud & Peksa, 2010), which are closely related to the

caulon lichens and the association between this diversity and envi-

genus Stereocaulon, has also been described. Both mycobiont genera,

ronmental conditions. This study represents the first investigation

Cladonia and Lepraria, associate with a wide range of Asterochloris

aimed at understanding the effects of climatic conditions, substrate/

species, which require diverse ecological conditions (Peksa & Ška-

habitat types, spatial structure and symbiotic partner (mycobiont) on

loud, 2011; Škaloud, Steinová, Řídká, Vančurová, & Peksa, 2015). In

the diversity of lichen phycobionts on a global scale. We applied

contrast, more diversified phycobionts in the microlichen genus

both phylogenetic and statistical analyses to numerous Stereocaulon

Micarea were found to associate with two genera, Coccomyxa and

specimens collected in diverse habitats worldwide to address the fol-

Elliptochloris (Trebouxiophyceae; Yahr, Florence, Škaloud, & Voyt-

lowing questions: (a) What is the diversity of phycobionts associated

sekhovich, 2015). A much broader range of potential photobiont

with the lichen‐forming genus Stereocaulon within the entire genus

partners was observed for species of the family Verrucariaceae,

and species‐level lineages? (b) Which environmental factors influence

where the mycobionts associate with phycobionts of nine genera in

the global distribution of phycobionts? (c) Do phycobionts and myco-

five orders of the Chlorophyta and one genus in Xanthophyceae

bionts exhibit reciprocal specificity/selectivity, and how does this

(Thüs et al., 2011).

affect the width of their climatic niches?

Stereocaulon (Lecanorales, Ascomycota) is a widely distributed, ecologically successful lichen‐forming genus, comprising mycobiont species with broad ecological requirements and extensive geographical distribution, sometimes associating with both phycobionts and cyanobionts (the latter located in particular structures known as cephalodia; Lücking et al., 2009). Stereocaulon lichens occur in highly

2 | MATERIAL AND METHODS 2.1 | Taxon sampling A total of 282 Stereocaulon specimens belonging to 20 fungal mor-

diverse environments, from polar (Seo et al., 2008) to tropical

phospecies (of 130–140 known morphospecies; Högnabba, 2006)

regions (Ismed et al., 2012), at different altitudes, and frequently on

collected all over the world (Figure 1, Supporting Information Table

metal‐rich substrates (Medeiros, Fryday, & Rajakaruna, 2014; Purvis

S1) were analysed. The following data were collected for the lichen

& Halls, 1996). Some species of this genus also tolerate submersion

samples: type of substrate, habitat, GPS coordinates and altitude.

(Sadowsky, Hussner, & Ott, 2012), as well as drought (Singh, Ranjan,

The sampling sites represented various habitats and diverse types of

Nayaka, Pathre, & Shirke, 2013). Stereocaulon ranks among the

substrates and were located at an altitude of 17–4,500 m

3018

|

VANČUROVÁ

ET AL.

F I G U R E 1 Distribution of phycobionts associating with the lichen‐forming fungal genus Stereocaulon. Blue dots—Asterochloris, red dots— Chloroidium, white dots—Vulcanochloris. Legend—annual mean temperature gradient. Magnified cut‐outs—(a) La Palma (Canary Islands), (b) Madeira, (c) Czech Republic

(Supporting Information Table S1). The sampling was carried out in

Fatehi, & Bridge, 1999). The algal and fungal nuclear internal tran-

2008–2016, and attempts were made for the sampling to be as com-

scribed spacer (ITS, ITS1‐5.8S‐ITS2 rDNA) and the algal actin type I

prehensive as possible concerning both the Stereocaulon morphos-

gene (including one complete exon and two introns located at codon

pecies and their ecology. The mycobiont morphospecies were

positions 206 and 248; Weber & Kabsch, 1994) were PCR amplified

identified using standard morphological and chemical analyses.

using primers listed in Supporting Information Table S2. The PCR

Chemical analyses involved thin‐layer chromatography (TLC) on

conditions were as follows: an initial denaturation at 94°C for 5 min

Merck silica gel 60 F254 precoated glass plates in solvent systems

followed by 35 cycles of denaturing at 94°C for 1 min, annealing at

A, B and C according to Orange, James, and White (2001). Lichen

50°C for 1 min and elongation at 72°C for 2 min, with a final exten-

specimens were deposited in the herbaria GZU, PL, PRA and PRC

sion step at 72°C for 10 min. The actin type I locus was amplified as

(herbaria acronyms follow Index Herbariorum; Thiers, 2016), and the

described by Peksa and Škaloud (2011) using four algal‐specific pri-

private herbarium of J. Malíček.

mer pairs (Supporting Information Table S2). All PCR amplifications were performed in a volume of 20 μl with Red Taq Polymerase (Sigma) as described by Peksa and Škaloud (2011) or with My Taq

2.2 | Phycobiont isolation, DNA extraction, amplification and sequencing

Polymerase. Negative controls, without DNA template, were

DNA was extracted from phycobiont cultures or directly from lichen

by contaminants in the reagents. The PCR products were purified

thalli (total lichen DNA). Phycobionts were isolated using the thallus

and sequenced using the same primers with an Applied Biosystems

fragment method (Ahmadjian, 1993) and cultivated as described in

(Seoul, Korea) automated sequencer (ABI 3730XL) at Macrogen in

Peksa and Škaloud (2008). Lichen thalli were examined under a dis-

Seoul, Korea. The newly obtained sequences of the ITS rDNA and

secting microscope and washed before DNA extraction to prevent

actin type I regions were deposited in GenBank under accession

contamination by soredia from other lichens. DNA was extracted

numbers MH382116–MH382150 and MH414969–MH415451 (Sup-

from thallus fragments following the CTAB protocol (Cubero, Crespo,

porting Information Table S1).

included in every PCR run to eliminate false‐positive results caused

VANČUROVÁ

|

ET AL.

maximum parsimony (MP) analysis using

2.3 | Sequence alignment and DNA analyses

PAUP

3019

v.4.0b10 (Swofford,

2003). BI and ML analyses were carried out on a partitioned data

Individual sequence alignments were prepared separately for Aste-

set to differentiate among ITS1, 5.8 S and ITS2 rDNA, actin intron

rochloris

considerable

206, actin intron 248 and actin exon regions. Substitution models

sequence divergence at the ITS locus. In addition, the sequences

and

Chloroidium

because

they

present

(Supporting Information Table S4) were selected using the Bayesian

obtained for Asterochloris were analysed as a single locus data set

information criterion (BIC) as implemented in JModelTest2 (Darriba,

for the ITS rDNA (data not shown) and as a concatenated data set

Taboada, Doallo, & Posada, 2012; Guindon & Gascuel, 2003). ML

of ITS rDNA and actin type I loci. The Vulcanochloris samples utilized

analysis was carried out using default settings, five search replicates

in this study were derived from the recent analysis of Vančurová et

and the automatic termination set at 5 million generations. The MP

al. (2015), and therefore, no new phylogenetic inference is presented

analysis was performed using heuristic searches with 1,000 random

here. Alignment of ITS rDNA sequences of Stereocaulon mycobionts

sequence addition replicates and random addition of sequences (the

was prepared.

number was limited to 10,000 per replicate). ML and MP bootstrap

The Asterochloris ITS rDNA data set consisted of 220 sequences:

support values were obtained from 100 and 1,000 bootstrap repli-

168 newly obtained sequences from Stereocaulon specimens and one

cates, respectively. Only one search replicate was applied for ML

newly obtained sequence from Cladonia, 19 previously published

bootstrapping.

sequences from Stereocaulon and 32 sequences from other lichens retrieved from GenBank. The actin type I data set consisted of 74 sequences: 31 newly obtained sequences from Stereocaulon speci-

2.4 | Species‐level lineages delimitation

mens, 11 previously published sequences from Stereocaulon and 32

We performed three species delimitation analyses (GMYC, bPTP,

sequences from other lichens. When selecting the available

ABGD) to estimate putative species boundaries in the Asterochloris,

sequences from GenBank, the care was taken to include all known

Chloroidium and Stereocaulon (mycobiont) data sets. As the presence

Asterochloris species as well as other previously published Asterochlo-

of identical sequences may result in artefactual species trees (Hoef‐

v.7

Emden, 2012), we merged all identical sequences in our data set.

ris species‐level lineages. The alignment was produced by

MAFFT

software (Katoh & Standley, 2013) under the Q‐INS‐I strategy and

First, we performed the Bayesian analyses with

manually edited according to the published secondary structures of

mond, Suchard, Xie, & Rambaut, 2012) to obtain ultrametric trees

ITS2 (Škaloud & Peksa, 2010) using

(Tamura, Stecher, Peter-

under the assumption of uncorrelated lognormal relaxed molecular

son, Filipski, & Kumar, 2013). The actin type I sequences were

clock. For each of the alignment partitions, the most appropriate

v.7 software (Katoh & Standley, 2013) under the

substitution model (Supporting Information Table S4) was estimated

Q‐INS‐I strategy. After deleting identical sequences, the resulting

using the Bayesian information criterion (BIC) as implemented in

concatenated alignment comprised 71 samples represented by 71

JModelTest2 (Darriba et al., 2012; Guindon & Gascuel, 2003). The

ITS rDNA (Supporting Information Appendix S1) and 66 actin type I

analyses were performed under the constant population size coales-

sequences (Supporting Information Appendix S2); missing actin data

cent as the tree prior and Ucld mean prior was set to exponential

were replaced with question marks.

distribution with mean 10 and initial value 1. Five MCMC analyses

aligned using

MAFFT

MEGA6

The Chloroidium ITS rDNA data set comprised 111 sequences:

BEAST

1.8.2 (Drum-

were run for 30 million generations, sampling every 10,000 generav.

80 newly obtained sequences from Stereocaulon specimens and 31

tions. The outputs were diagnosed for convergence using

representative sequences from all known free‐living Chloroidium spe-

1.7 (Rambaut, Drummond, Xie, Baele, & Suchard, 2018), and the five

cies (Supporting Information Table S3). The alignment was produced

tree files were merged using the burn‐in set to 3 million generations

v.7 software (Katoh & Standley, 2013) under the Q‐INS‐I

(all ESS values of the merged data set were above 900). Consensus

by

MAFFT

strategy and manually edited using

MEGA6

(Tamura et al., 2013)

according to the ITS2 secondary structures constructed by RNAfold

TRACER

tree was generated using TreeAnnotator 1.8.2. The GMYC analysis was performed on ultrametric consensus tree

WebServer (Gruber, Lorenz, Bernhart, Neuböck, & Hofacker, 2008)

under the single‐threshold model, using the

with default settings. After removing identical sequences, the result-

aghan et al., 2009) in

ing alignment comprised 45 sequences (Supporting Information

sis was also performed on ultrametric consensus tree, using the

Appendix S3).

bPTP web Server (http://species.h-its.org/ptp/). The analysis was run

R

SPLITS

package (Mon-

3.3.0 (R Core Team, 2017). The bPTP analy-

The Stereocaulon mycobiont ITS rDNA data set consisted of 335

for 200,000 generations, using 0.3 burn‐in and 100 thinning. Both

sequences: 234 newly obtained sequences from our Stereocaulon

ML and Bayesian solutions were examined. At last, the ABGD analy-

specimens and 88 previously published sequences. The alignment

sis was performed on the concatenated alignment, using the ABGD

v.7 software (Katoh & Standley, 2013)

web server (http://wwwabi.snv.jussieu.fr/public/abgd/abgdweb.html).

under the Q‐INS‐I strategy. After removing identical sequences, the

Genetic distances were calculated using the K80 model, and the

was produced using

MAFFT

resulting alignment comprised 195 sequences (not presented).

model parameters were set to Pmin 0.001, Pmax 0.01, Steps 10 and

Phylogenetic trees were inferred with Bayesian Inference (BI)

Nb bins 20. Separate analyses were run under varying relative gap

using MrBayes v.3.2.2 (Huelsenbeck & Ronquist, 2001), maximum‐

width values (0.1, 0.3, 0.5, 0.8, 1.0) to assess the consistency of the

likelihood (ML) analysis using

GARLI

v.2.0 (Zwickl, 2006), and

inferred groups.

3020

|

VANČUROVÁ

ET AL.

A name was assigned to the recovered lineages of Asterochloris

species niche as an n‐dimensional hypervolume, where the dimen-

and Chloroidium using (a) the original name given to the lineage

sions are environmental variables (Hutchinson, 1957). In this study,

when it was first published (e.g., A9); (b) the name of a known spe-

these environmental dimensions were described based on 19 Bioclim

cies that had been formally described in previous phylogenetic

variables (Karger et al., 2017). The climatic hypervolumes were con-

studies; (c) the names A. aff. irregularis, A. aff. italiana and C. aff. ellip-

structed by multivariate kernel density estimation (Blonder, Lamanna,

soideum indicating affinity to that species; or (d) the nomenclature

Violle, & Enquist, 2014). First, we performed the PCA analysis of 19

StA1–StA8 (Asterochloris) and StC1 and StC2 (Chloroidium) to indicate

Bioclim variables to reduce the total number of predictors. First, two

lineages identified as new and not yet formally described. For the

PCA axes (explaining 65% of the total variance) were then selected

algal species‐level lineages containing only one sample, we used the

to calculate hypervolumes for each species‐level lineages and genera.

name of that sample (e.g., sample A504). For this study, the species‐

The boundaries of the kernel density estimates were delineated by

level lineages of Stereocaulon mycobiont were named OTU1–OTU57.

the probability threshold, using the 0.85 quantile value. To project

The taxonomic revision of Stereocaulon is not the aim of this study,

the niche spaces of particular lineages, hypervolume contours were

and therefore, the species names (S. vesuvianum, S. azoreum and

plotted based on 5,000 random background points, using the alpha-

S. nanodes) were assigned to the lineages only when their identity

hull contour type and alpha smoothing value 0.55. The analyses

was obvious.

were performed in R, using the hypervolume (Blonder et al., 2014) and alphahull (Pateiro‐Lopez & Rodriguez‐Casal, 2016) packages. The relationship between specificity towards the symbiotic part-

2.5 | Variation partitioning

ner and width of climatic niche was inspected as correlation

From the entire data set of 282 Stereocaulon specimens, 35 were

between the number of accepted partners and size of climatic hyper-

excluded due to the lack of mycobiont sequences and 6 due to the

volume. As the number of samples of particular species‐level lineages

insufficient substrate/habitat data, resulting to a data set of 241

varied significantly, the number of accepted species‐level lineages of

samples. The relative effects of climate, substrate/habitat, geographi-

symbiotic partners were down‐sampled to the smallest sample size

cal distance and the symbiotic partner on the variance in photobiont

in the data set, which is 15 samples for the seven most abundant

as well as mycobiont diversity were analysed by variation partition-

lineages of mycobiont and 11 for eight most abundant lineages of

ing in redundancy analysis, using the varpart function in the vegan

phycobiont (Supporting Information Figure S1). The rarefaction was

package (Oksanen et al., 2017). The phylogenetic distances of phy-

performed using rarefy function in vegan package (Oksanen et al.,

cobionts or mycobionts were used as a response variable, coded as

2017). The linear regression was performed separately for the myco-

the first 10 PcoA axes. Climatic data were obtained from the

biont and phycobiont species‐level lineages. As the parametric

CHELSA Bioclim database (Karger et al., 2017) at a resolution of 2.5

regression analyses can be significantly biased in small sample sizes,

arc minutes. At each sampling site, climatic data were obtained by

we performed the Bayesian linear regression instead. We con-

applying a 5 km buffer to limit the effects of spatial bias. The 19

structed a regression model where we modelled the number of

environmental variables were condensed into principal component

accepted species‐level lineages (Xi) as Xi ∼ Normal (μi, σ), where μi

variables (PCs). The Broken‐stick distribution (Jackson, 1993) was

was determined as a + b * hypervolumei (a = intercept, b = slope of

used to select which principal components to include in variation‐

the regression line) and σ as the variance of the residuals. The priors

partitioning analysis, using the bstick function in the vegan package

were set as follows: a ∼ Normal (0, 0.001), b ∼ Normal (0, 0.001),

(Oksanen et al., 2017). Therefore, PC1–PC4 were selected. Based on

σ ∼ Uniform (0, 100). We ran three chains of the model for

an analogy, the presence/absence matrix of 12 substrate/habitat vari-

1,000,000 iterations, discarding the initial 100,000 as burn‐in. We fit

ables (Supporting Information Table S5) was transformed into princi-

the regression model in program

pal component variables. Again, PC1–PC4 were selected by the

the R2JAGS package in R.

JAGS

4.2.0 (Plummer, 2003) through

Broken‐stick distribution. Geographical distance values (latitude and longitude) were transformed to the principal coordinates of neighbour matrices (PCNM) vectors representing the geographical distances at various spatial scales (Borcard, Legendre, Avois‐Jacquet, & Tuomisto, 2004). PCNM vectors were calculated based on the pairwise geographical distances obtained by the distGPS function in the BoSSA package (Lefeuvre, 2018). The first 100 PCNM were used for the analysis. All analyses were performed in

R

(R Core Team, 2017).

3 | RESULTS 3.1 | Molecular sequence data and phylogenetic analysis In total, we generated 518 new sequences, which were deposited in GenBank under accession numbers MH382116–MH382150 and MH414969–MH415451 (Supporting Information Table S1), and the

2.6 | Niche hypervolumes

alignments have been deposited as Supporting Information Appendices S1–S3.

The climatic niche of the most abundant species‐level lineages of

Based on their ITS rDNA sequence analysis, the phycobionts in

phycobionts and mycobionts and three genera of phycobionts were

Stereocaulon belong to three genera: Asterochloris, Chloroidium and

represented using the Hutchinsonian niche concept that describes a

Vulcanochloris. Asterochloris and Vulcanochloris are closely related

VANČUROVÁ

|

ET AL.

3021

genera within the order Trebouxiales, whereas Chloroidium belongs

detected in three species: V. canariensis, V. symbiotica and V. guan-

to the unrelated Watanabea clade within the same class, Trebouxio-

chorum. In several cases, we recovered more than one phycobiont geno-

phyceae. The phylogenetic hypothesis resulting from Bayesian analysis of

type from a single lichen thallus (either by direct sequencing of total

the ITS rDNA and actin type I sequences of Asterochloris is shown in

DNA or by genotyping multiple cultures isolated from a single thallus).

Supporting Information Figure S2. We recovered phylogenetic rela-

Representatives of Chloroidium ellipsoideum and C. angustoellipsoideum

tionships congruent with those obtained in previous studies (Moya

were detected simultaneously four times (samples VancurovaA421,

et al., 2015; Peksa & Škaloud, 2011; Škaloud et al., 2015). According

VancurovaLV5, VancurovaOP1118 and VancurovaOP1083; up to

to three DNA species delimitation analyses (GMYC, bPTP and

six sequences from a single lichen sample). Three sequences of

ABGD), putative species boundaries in Asterochloris data set were

sample VancurovaOP1077 (Vancurova1077, VancurovaOP1077.1

estimated. The species were delimited based on the consensus of

and

VancurovaOP1077.2)

correspond

to

three

divergent

these analyses, leading to the delimitation of 39 species clusters. We recovered sequences from Stereocaulon thalli in 27 lineages, 10 of which (lineages Asterochloris aff. irregularis, A. aff. italiana and StA1‐ StA8) are new highly resolved lineages in Asterochloris. The majority of the new lineages exclusively comprise newly obtained sequences,

mycobiont 47 %

whereas others include previously published sequences with unresolved positions in Asterochloris phylogenetic analyses in previous studies (Cordeiro et al., 2005; Moya et al., 2015; Peksa & Škaloud,

climate

2011; Piercey‐Normore & DePriest, 2001; Škaloud & Peksa, 2010). Nine of the Asterochloris lineages could be assigned to formally described species, namely A. erici, A. excentrica, A. glomerata, A. ital-

7%

geography 7%

2%

iana, A. irregularis, A. lobophora, A. mediterranea, A. phycobiontica and

21 %

A. woessiae. The most frequently occurring phycobionts belonged to the species A. irregularis, accounting for 32% of Asterochloris phycobionts of Stereocaulon. The phylogenetic backbone sustains the three

3% 1% substrate 2%

main clades, clades A–C, sensu Škaloud and Peksa (2010). Even

Residuals = 8 %

though the phycobionts of Stereocaulon were recovered in all three Asterochloris clades, they differed in the abundance of Stereocaulon sequences; clade B includes only 16 of these sequences, whereas 103 and 68 sequences were recovered within clades A and C, respectively. A phylogram resulting from Bayesian analysis of ITS rDNA

6%

F I G U R E 2 Venn's diagram showing the variation in distribution of phycobionts associated with the lichen‐forming fungal genus Stereocaulon explained by effects of climate, substrate/habitat, geographical distance and the mycobiont [Colour figure can be viewed at wileyonlinelibrary.com]

sequences of Chloroidium is shown in Supporting Information Figure S3. The phylogenetic relationships are congruent with those identified by Darienko et al. (2010). According to the three DNA species delimitation analyses (GMYC, bPTP and ABGD), putative species boundaries in Chloroidium data set were estimated. The species were delimited based on the consensus of these analyses, leading to the delimitation of 12 species clusters. The Chloroidium

phycobiont 42 % geography

phycobionts analysed were clustered into nine lineages. Two of the lineages could be placed in formally described species, C. ellipsoideum

2%

and C. angustoellipsoideum, whereas StC1, StC2 and C. aff. ellipsoideum are new lineages in Chloroidium (clade StC2 contains one new and one previously published sequence). Three of the nine lin-

3%

1%

54% of Chloroidium phycobionts of Stereocaulon. In contrast, representatives of C. saccharophilum and C. engadiensis were not found to be phycobionts. The Vulcanochloris data set was previously analysed by Vančurová et al. (2015). The phycobionts belonging to this genus were recovered in 15 Stereocaulon thalli. All identified ITS sequences were

2%

5%

eages also include free‐living algae. The most frequently occurring phycobionts belong to the species C. aff. ellipsoideum, accounting for

2% 3%

climate

10 %

substrate 3%

7% Residuals = 24 % F I G U R E 3 Venn's diagram showing the variation in distribution of Stereocaulon mycobionts explained by effects of climate, substrate/habitat, geographical distance and the phycobiont [Colour figure can be viewed at wileyonlinelibrary.com]

|

OTU52

OTU9 sample A316

ET AL.

V. symbiotica

OTU11

OTU43 OTU57 OTU27 OTU26 OTU44 A. mediterranea sample A14 StC1 sample A503 StA6 V. canariensis V. guanchorum

C. ellipsoideum

A9

OTU18 OTU23 OTU32 OTU42

C. aff. ellipsoideum

StC2

C. angustoellipsoideum

OTU40 S. nanodes

OTU10 S. vesuvianum StA1

sample A318 sample A504

OTU45 OTU49 OTU31 A. irregularis

OTU47

OTU29 A. glomerata

OTU50 A. aff. irregularis

A. italiana

A. lobophora

A. woessiae

OTU12 OTU37 OTU3

OTU13 S. azoreum

OTU35

StA2 A. phycobiontica StA8 Clade 12 A. aff. italiana

StA5

StA4 Clade 8

OTU22

VANČUROVÁ

OTU34 OTU36 OTU38 OTU39

3022

F I G U R E 4 Interaction network structure between lichen mycobiont species‐level lineages in the genus Stereocaulon and phycobiont species‐level lineages. The width of the links is proportional to the number of specimens forming the association [Colour figure can be viewed at wileyonlinelibrary.com] C. ellipsoideum

Van-

(2006). Many morphospecies in both our and Högnabba's phylo-

curovaKO25.1 and VancurovaKO25.2 classified into two genera,

genotypes.

Moreover,

the

sequences

gram were paraphyletic, but some lineages clearly correspond

Asterochloris and Chloroidium, respectively.

with morphospecies. According to three DNA species delimitation

In contrast, multiple sequences from a single sample were often

analyses (GMYC, bPTP and ABGD), putative species boundaries

identical; VancurovaL1248 (direct from thallus) and DS1.1 (from cul-

in the Stereocaulon mycobiont data set were estimated. The spe-

ture) represent Asterochloris irregularis, and sequences L952 (direct

cies were delimited based on the consensus of different analyses,

from thallus) and CAB.1 and CAB.2 (from culture) are from the same

leading to the delimitation of 57 species clusters. We recovered

genotype of C. ellipsoideum.

sequences from Stereocaulon thalli in 30 lineages. The most fre-

The phylogenetic hypothesis resulting from Bayesian analysis

quently occurring mycobionts belonged to OTU10, which corre-

of the ITS rDNA sequences of Stereocaulon mycobionts (not

sponds with species S. vesuvianum/S. arcticum, accounting for

shown) is largely congruent with that identified by Högnabba

24% of samples.

F I G U R E 5 Climatic niche hypervolumes for (a) algal genera Asterochloris, Vulcanochloris and Chloroidium, (b) eight most abundant algal species‐level lineages (phycobionts), (c) seven most abundant fungal species‐level lineages (mycobionts), (d) fungal OTU10 (grey filled) with its seven most abundant (of total 11) associating phycobionts, (e) fungal OTU35 (grey filled) with its seven most abundant (of total 12) associating phycobionts based on climatic PC1–PC2 axes (explaining 65% of variation). Climatic variables: 1 = annual mean temperature, 2 = mean diurnal range, 3 = isothermality, 4 = temperature seasonality, 5 = max temperature of warmest month, 6 = min temperature of coldest month, 7 = temperature annual range, 8 = mean temperature of wettest quarter, 9 = mean temperature of driest quarter, 10 = mean temperature of warmest quarter, 11 = mean temperature of coldest quarter, 12 = annual precipitation, 13 = precipitation of wettest month, 14 = precipitation of driest month, 15 = precipitation seasonality, 16 = precipitation of wettest quarter, 17 = precipitation of driest quarter, 18 = precipitation of warmest quarter, 19 = precipitation of coldest quarter (Karger et al., 2017)

VANČUROVÁ

|

ET AL.

Climatic variables − PCA

Asterochloris

1.0 1718

13 16

Precipitation in warm and wet periods

19

de

3

alt

itu

Precipitation in cold and dry periods Temperature in cold and dry periods 11 9 6

2

0.0 7 4

5

0.5

Chloroidium

12

Extremes in temperatures

0

14

1 evapotranspiration 15

−0.5 Temperature in warm 8 5 and wet periods

10

−0.5

0.5

−5

Dim2 (27.8%)

(a)

Vulcanochloris

−1.0 0.0 Dim1 (37.5%)

A. irregularis A. woessiae Asterochloris StA1 Asterochloris StA5 C. ellipsoideum C. aff. ellipsoideum C. angustoellipsoideum V. symbiontica

1.0

−4

–6

OTU10 OTU11 OTU13 OTU35 OTU40 OTU47 OTU52

0

2

4

6

(c)

−4

−5

−2

0

0

2

(b)

−2

4

−1.0

−6

−4

OTU10 (mycobiont) A. aff. irregularis A. irregularis A. glomerata A. italiana Asterochloris A9 Asterochloris StA1 C. aff. ellipsoideum

−2

0

2

4

6

−8

−6

OTU35 (mycobiont) A. aff. irregularis A. lobophora A. woessiae Asterochloris StA8 Asterochloris StA2 Asterochloris StA4 Asterochloris StA5

0

2

4

(e)

−4

−6

−4

−2

−2

0

0

2

2

(d)

−2

−4

−6

−4

−2

0

2

4

6

−5

0

5

6

3023

3024

|

3.2 | The associations between phycobiont, mycobiont and environmental conditions

VANČUROVÁ

ET AL.

Among the algal genera (Figure 5a), Asterochloris and Chloroidium have relatively wide niches, unlike Vulcanochloris. The climatic data suggest that Asterochloris prefers humid climates, Vulcanochloris tol-

To identify the factors that shape the symbiotic partner distribution

erates extremely dry conditions, and Chloroidium accepts a wide

of Stereocaulon lichens, we performed variation‐partitioning analyses

range of humidity levels (Figure 6a). We also detected obvious dif-

(Figures 2 and 3). We analysed the relative contributions of climate,

ferences in precipitation seasonality (Figure 6b): Asterochloris occurs

habitat/substrate, geographical distance and symbiotic partner to

in conditions with the most stable precipitation levels, whereas

phycobiont and mycobiont distribution.

Chloroidium accepts highly variable precipitation levels. Asterochloris

Among the phycobionts, climatic conditions, substrate and habitat,

seems to be the most psychrophilic of the three genera, unlike Vul-

geographical distance and the symbiotic partner (i.e., mycobiont)

canochloris, which likely prefers relatively elevated temperatures. In

explained 92% of the variation (Figure 2). The largest proportion of

conclusion, Chloroidium phycobionts were found at an annual mean

the variation was explained by the mycobiont (47% independent effect

temperature above 0°C (Figure 6c). Only one exception of this rule

and 22% in combination with other variables). Several algal species‐

was observed: Sample VancurovaA35 was found at a location with

level lineages showed specificity towards a single mycobiont OTU (al-

an annual mean temperature of −2°C.

gal‐fungal pairs StA1‐OTU10 and V. symbiotica‐OTU52; Figure 4).

As represented on the plot of the hypervolumes of the eight

Others were not specific towards a single mycobiont, but co‐operate

most abundant phycobionts (Figure 5b), the climatic niche of the

in most cases with one fungal species‐level lineage (i.e., it is selective

genus Asterochloris is composed of quite distinct niches of species‐

towards symbiotic partner). For example, OTU47 accepts three algal

level lineages. The climatic data suggest that algal species‐level lin-

species‐level lineages, but prefers A. irregularis (Figure 4). Geographical

eages are quite heterogeneous in terms of temperature preference

distance independently explained 7% of the variability, although 33%

(Figure 7). It is an interesting fact that Asterochloris italiana and

was shared with other variables. The variables associated with sub-

A. woessiae appear to be relatively thermophilic within the generally

strate and habitat independently explained 1% of the variability,

psychrophilic genus. These two lineages also occur in more stable

although 13% was shared with other variables. The climatic conditions

climates, as distinct from lineage StA5 and A. irregularis, which seem

explained 33% of the variability shared with other variables (21% with

to tolerate considerable temperature seasonality (Figure 5b; Support-

geography), but explained nothing independently.

ing Information Figure S4).

Climatic conditions, substrate and habitat, geographical distance

The seven most abundant mycobiont species‐level lineages could

and the symbiotic partner (i.e., phycobiont) explained 76% of the

be divided into specialists or generalists with narrow or broad cli-

variation in the phylogeny of mycobionts. The greatest proportion

matic niches, respectively (Figure 5c). They also differ in their speci-

of the variation (42% independent effect, 15% shared with other

ficity towards their algal partner (3–12 algal partners within the

variables) was explained by the symbiotic partner, analogically.

entire data set and 2.8–8.6 algal partners after down‐sampling to the

Although all largely represented mycobiont species‐level lineages

smallest sample size in the data set). The similar pattern was also

co‐operate with several species of phycobionts, at the level of

observed within the eight most abundant phycobiont species‐level

algal genera they are mostly specific (Figure 4). Geographical dis-

lineages, which co‐operate with 1–8 fungal partners (1–4.59 fungal

tance was the second most important variable, which indepen-

partners after the down‐sampling). The hypothesis that species with

dently explained 10% of the variability, although 11% was shared

wide niches corroborate with more symbiotic partners was con-

with other variables. Besides worldwide distributed mycobionts

firmed using the Bayesian linear regression for fungal as well as algal

(especially OTU10), species‐level lineages with limited distribution

species‐level lineages (Figure 8; Supporting Information Figures S5

were also identified. For example, OTU52 was found only on La

and S6). For two fungal species‐level lineages with the widest cli-

Palma island (Canary Islands), OTU11 as well as OTU13 (S. azor-

matic niches (OTU10 and OTU35) and the most algal partners, plots

eum) in the Mediterranean region, and OTU47 in the Circumboreal

combined fungal hypervolume with hypervolumes of their phyco-

region. The climatic variables independently explained 7% of the

bionts were produced (Figure 5d,e).

variability, although 16% was shared with other variables. The fourth variable, substrate and habitat characteristics, accounted for only a small proportion of the variation (3% of the independent effect, 10% in combination with other variables).

3.3 | Climatic niches and specificity between the symbiotic partners

4 | DISCUSSION 4.1 | Phycobiont diversity This study provides insights into the genetic diversity and ecological requirements of phycobionts associated with the lichen‐forming fungal genus Stereocaulon worldwide. In Stereocaulon, the main phyco-

We constructed two‐dimensional (PC1‐PC2 explaining 65.3% varia-

biont genus is Asterochloris, for which we recovered 27 lineages

tion of climatic variables) hypervolumes for seven most abundant

(Supporting Information Figure S2), although the second and the

fungal species‐level lineages, three algal genera and the eight most

third most prevalent genera are Chloroidium and Vulcanochloris,

abundant algal species‐level lineages.

respectively.

(a)

|

ET AL.

Precipitaon of driest quarter (mm)

3025

Annual Mean temperature(°C) 20 15

800

1,000

VANČUROVÁ

600

10

V. symbioca

C. ellipsoideum

C. aff. ellipsoideum

C. angustoellipsoideum

Asterochloris StA5

Asterochloris StA4

Asterochloris StA1

A. woessiae

A. lobophora

A. italiana

Asterochloris A9

Precipitaon seasonality (%)

A. irregularis

Vulcanochloris

80

(b)

Chloroidium

A. glomerata

Asterochloris

A. aff. irregularis

400

–5

0

0

200

5

40

60

F I G U R E 7 Differences in the distribution of 14 most abundant (≥5 specimens) phycobiont species‐level lineages associated with the lichen‐forming fungal genus Stereocaulon along the gradient of annual mean temperature

The phycobiont diversity observed here in Stereocaulon appears to be exceptional, especially in terms of the number of algal genera.

20

Lichens generally associate with multiple lineages belonging to a single photobiont genus. Indeed, a wide range of Trebouxia lineages are phycobionts of species belonging to various lichen genera, for example, Protoparmelia, Rhizoplaca, Tephromela, Xanthoparmelia, Xanthoria and

Asterochloris (c)

Chloroidium

Vulcanochloris

Annual Mean temperature (°C)

Xanthomendoza (Leavitt et al., 2013, 2016; Muggia et al., 2014; Muggia, Leavitt, & Barreno, in press; Nyati et al., 2013), and high infrageneric diversity of Asterochloris phycobionts has also been observed

20

in species of Cladonia (Bačkor et al., 2010; Beiggi & Piercey‐Normore, 2007; Piercey‐Normore & DePriest, 2001; Škaloud & Peksa, 2010; Yahr et al., 2004) and Lepraria (Nelsen & Gargas, 2006, 2008; Peksa &

10

Škaloud, 2011; Škaloud & Peksa, 2010). It is an interesting fact that only a few other lichens, which have crustose growth and (generally) a poorly developed cortex (Helms, 2003; Thüs et al., 2011), are known to build their thalli with phycobionts belonging to different Trebouxio-

0

phycean genera (Lepraria borealis, Engelen, Convey, & Ott, 2010; Micarea, Yahr et al., 2015; Bagliettoa and Verrucaria nigrescens, Thüs et

–10

al., 2011; Voytsekhovich & Beck, 2015; Diploschistes muscorum, Wedin et al., 2015). In contrast, the Stereocaulon species considered in this study have complex dimorphic thalli and a well‐developed cortex (crustose species of Stereocaulon were not included).

Asterochloris

Chloroidium

Vulcanochloris

F I G U R E 6 Differences in the distribution of three phycobiont genera associated with the lichen‐forming fungal genus Stereocaulon along the gradient of (a) precipitation of driest quarter; (b) precipitation seasonality; (c) annual mean temperature

Our results also expand upon the known diversity of Chloroidium in lichens, as three novel lineages were here identified (Supporting Information Figure S3) and four samples from Central America probably represent still undescribed species within Chloroidium. Also, Sanders, Pérez‐Ortega, Nelsen, Lücking, and de los Ríos (2016)

|

VANČUROVÁ

ET AL.

5

the ITS rDNA, we did not further analyse it here. Although previously

(a)

overlooked, the co‐occurrence of several phycobionts in individual

4

lichen thalli (i.e., algal plurality) is a relative common phenomenon (Bačkor et al., 2010; Moya, Molins, Martinez‐Alberola, Muggia, & Bar-

3

reno, 2017; Muggia, Baloch, Stabentheiner, Grube, & Wedin, 2011; Muggia et al., 2014; Onuț‐Brännström et al., 2018; Park et al., 2015;

2

Voytsekhovich & Beck, 2015). We also obtained evidence for algal plurality in several Stereocaulon samples, which strengthens the potential of this lichen genus as a suitable model for high‐throughput sequencing studies.

1

Number of fungal species-level lineages

3026

The phycobiont diversity in Stereocaulon should not be regarded

0

10

20

30

40

50

only from a taxonomic or systematic point of view; instead, it also

60

Niche space of phycobiont

extends to the different ecological requirements of the phycobionts

9

(Álvarez et al., 2012; Casano et al., 2011; Del Hoyo et al., 2011),

(b)

8

also in Stereocaulon the co‐occurrence of phycobionts with diverse physiological responses could be an effective adaptive strategy for

7

the successful, pioneering colonization of habitats.

6

In contrast to lichen symbioses, algal plurality for coral ecosystems has been explored in greater detail. Several studies have sug-

5

gested the co‐occurrence of multiple Symbiodinium lineages within

4

individual hosts (Baker, 2003; Baums, Devlin‐Durante, & Lajeunesse,

3

2014). Particular lineages of Symbiodinium show distinct ecological preferences (Baker, 2003; Pettay, Wham, Smith, Iglesias‐Prieto, &

2

Number of algal species-level lineages

involved (see below). As demonstrated for Ramalina farinacea

LaJeunesse, 2015; Rowan, 2004), and some are well adapted to high temperatures and irradiance (Iglesias‐Prieto, Beltrán, LaJeunesse,

0

10

20

30

40

50

Niche space of mycobiont F I G U R E 8 (a) Bayesian linear regression of algal niche space (hypervolume) as a predictor of the number of accepted species‐ level fungal lineages. (b) Bayesian linear regression of fungal niche space (hypervolume) as a predictor of the number of accepted species‐level algal lineages. Dashed lines show the 95% CRI around the regression line [Colour figure can be viewed at wileyonlinelibrary.com]

Reyes‐Bonilla, & Thomé, 2004). The ability of corals to maintain or switch various algae could be influenced by the diversity of possible symbionts, which varies among areas (Baums et al., 2014). However, although juvenile corals maintain several strains, or switch strains frequently (Byler, Carmi‐Veal, Fine, & Goulet, 2013), the capacity of adult corals to switch photobionts is rather limited (Baums et al., 2014; Byler et al., 2013; Iglesias‐Prieto et al., 2004). It is therefore necessary to clarify whether the aforementioned phycobiont co‐ occurrences in the Stereocaulon species are as stable as that of the

recently presented a new lineage of phycobiont sister to the Chloroid-

pair Trebouxia jamesii/Trebouxia TR9 found in Ramalina farinacea, or

ium clade from the lichen Bapalmuia lineata, which grows on leaves in

whether they represent only transitional phases of algal switching

Panama, which suggest Central America to host an unexplored

(Wedin et al., 2015).

diverse group of symbiotic algae. In general, the genus Chloroidium has rarely been reported in lichens (Beck, 2002), being known only from the genera Trapelia (Beck, 2002; Tschermak‐Woess, 1948,

4.2 | Ecology and distribution of phycobionts

1978), Psilolechia, Lecidea (Beck, 2002), Bacidia (Tschermak‐Woess,

Our results suggest that amount and seasonality of precipitation

1988a), Verrucaria (Voytsekhovich & Beck, 2015), Galidea and Gom-

may be key factors affecting the distribution of the three phyco-

phillus (Sanders et al., 2016). By bad luck, most of these reports can-

biont genera (Figure 6a,b). According to climatic data, the distribu-

not be compared with our results, because the studies were based

tion of Vulcanochloris as a phycobiont of Stereocaulon is restricted

mainly on morphology, and little molecular data were published. Only

to areas with precipitation during the driest quarter, ranging from

the recent work of Sanders et al. (2016) offers rbcL sequences com-

3 to 6 mm. Chloroidium occurs in areas with a broad range of pre-

parable to those generated by our group. The sequence of the phyco-

cipitation during the driest quarter (4–960 mm), whereas Aste-

biont of Galidea (KX235274; Sanders et al., 2016) is identical to the

rochloris is distributed in areas with precipitation in the driest

rbcL sequence (not shown) of our sample VancurovaO24 collected in

quarter ranging from 6 to 316 mm. In terms of temperature vari-

New Zealand (Chloroidium aff. ellipsoideum), and the phycobiont of

ables (Figure 6c), Vulcanochloris appears to be the most ther-

Gomphillus (KX235269) appears to be a member of the StC2 lineage.

mophilic phycobiont (annual mean temperature up to 19.8°C) of

However, as the rbcL marker generally shows lower resolution than

Stereocaulon.

The

overwhelming

majority

of

Chloroidium

VANČUROVÁ

|

ET AL.

Number of samples

Geography

Type of habitat

3027

Substrate

A. glomerata A. irregularis A. aff. irregularis A. erici StA1 VancurovaA504 S1 clade8 StA2 S3 A. woessiae StA3 RidkaT20 A. mediterranea clade12 VancurovaA14 A. aff. italiana A. italiana StA4 A. phycobionca StA5 A. lobophora A. excentrica A9 StA6 StA7 StA8 0

70 0%

50%

100% 0%

50%

100% 0%

50%

100%

0

70 0%

50%

100% 0%

50%

100% 0%

50%

100%

0

70 0%

50%

100% 0%

50%

100% 0%

50%

100%

C. ellipsoideum C. aff. ellipsoideum StC1 VancurovaA316 StC2 VancurovaA318 VancurovaA419 C. angustoellipsoideum VancurovaA503 V. symbioca V. canariensis V. guanchorum

holarcs holantarcs paleotropis neotropis

anthropogenic natural unknown

volcanic rock other rock soil other or unknown

F I G U R E 9 Visualized abundance, geographic and habitat, and substrate distribution of phycobionts associating with the lichen‐forming genus Stereocaulon. From left to right: phylogenetic hypotheses based on ITS rDNA + actin type I gene (Asterochloris) or sole ITS rDNA sequences (other two genera); barcharts showing the absolute phycobiont abundances; proportional abundances in phytogeographical regions; proportional abundances in habitats; and proportional abundances on substrates

phycobionts are distributed in areas with an annual mean temper-

however, represented by only one sample in our data set. The

ature above 0°C. Although most of the Asterochloris species are

distribution of this species is concentrated in the Mediterranean

rather psychrophilic, A. italiana and A. woessiae prefer annual mean

region (Moya et al., 2015). It is not clear whether Asterochloris dis-

temperatures above 5°C and 10°C, respectively (Figure 7). Our

tribution is restricted by low temperatures or if the reduced

results complement the finding of Peksa and Škaloud (2011) who

amount of liquid water prevents its distribution in polar regions

showed that the genus Lepraria harbours A. woessiae phycobionts

(Engelen et al., 2010; Park et al., 2015). An example of the joint

at low altitudes in central and southeastern Europe. Another ther-

influence of temperature and humidity is as follows: in samples

mophilic lineage of this genus is A. mediterranea, which is,

from Alaska and Greenland, at very low temperatures (year mean

3028

|

VANČUROVÁ

ET AL.